1. Prepare a 0.1% trypsin solution in high-calcium HL saline. Aliquot and store at -20°C.
2. Prepare at least four fire-polished glass pipettes by briefly placing the tips of the pipettes in a flame.
3. Prepare melted 1% agar in high-calcium HL saline. Prepare at least four agar dishes by pouring the agar into culture dishes.
4. Thaw the trypsin solution immediately before use and add 1/100 volume of 1 M DTT solution to the trypsin solution.
5. Fill one agar dish with the trypsin/DTT solution and the other three with high-calcium HL saline (these will be the washing agar dishes).
6. Place embryos into the trypsin/DTT solution; watch them closely. The vitelline envelope should start to wrinkle, break open, and lift off from the embryo within a few minutes. The time required for each embryo varies significantly and ranges from 1 to 30 min. Use a fire-polished glass pipette to transfer an individual embryo to a washing agar dish containing high-calcium HL saline as soon as its vitelline envelope breaks loose.
7. Often, the vitelline envelope does not completely detach from the embryo. If this is the case, use a flame-polished micropipette (see Embryo Culture section for details) to remove the loosened vitelline envelope once the embryo is in the first washing agar dish.
8. Once the vitelline envelope is removed from every embryo, transfer the embryos to a new washing agar dish with a new flamed glass pipette. Incubate for 5 min.
9. Repeat Step 8 twice.
10. Transfer the embryos back to HL saline supplemented with antibiotics. They are now ready for further experimentation.

Note: Devitellinized embryos are fragile and sticky and should always be kept on an agar bed. The devitellinized embryos should be moved to a new agar dish containing fresh HL saline and antibiotics daily if long-term culture is desired.

Recipe

High-Calcium HL medium

NaCl (4.8 mM), KCl (1.2 mM), MgCl2 (2 mM), CaCl2 (16 mM), maleic acid (1 mM), pH 8

To make 1 L,

fill with ddH2O to 1 L,

adjust pH with 1 M NaOH (carefully and slowly; do not overshot; remake the solution if overshot the pH).